Recreating Stable Brachypodium hybridum Allotetraploids by Uniting the Divergent Genomes of B. distachyon and B. stacei.

Brachypodium hybridum (2n = 30) is a natural allopolyploid with highly divergent sub-genomes derived from two extant diploid species, B. distachyon (2n = 10) and B. stacei (2n = 20) that differ in chromosome evolution and number. We created synthetic B. hybridum allotetraploids by hybridizing various lines of B. distachyon and B. stacei. The initial amphihaploid F1 interspecific hybrids were obtained at low frequencies when B. distachyon was used as the maternal parent (0.15% or 0.245% depending on the line used) and were sterile. No hybrids were obtained from reciprocal crosses or when autotetraploids of the parental species were crossed. Colchicine treatment was used to double the genome of the F1 amphihaploid lines leading to allotetraploids. The genome-doubled F1 plants produced a few S1 (first selfed generation) seeds after self-pollination. S1 plants from one parental combination (Bd3-1×Bsta5) were fertile and gave rise to further generations whereas those of another parental combination (Bd21×ABR114) were sterile, illustrating the importance of the parental lineages crossed. The synthetic allotetraploids were stable and resembled the natural B. hybridum at the phenotypic, cytogenetic and genomic levels. The successful creation of synthetic B. hybridum offers the possibility to study changes in genome structure and regulation at the earliest stages of allopolyploid formation in comparison with the parental species and natural B. hybridum.

Prevalence of gene expression additivity in genetically stable wheat allohexaploids.

The reprogramming of gene expression appears as the major trend in synthetic and natural allopolyploids where expression of an important proportion of genes was shown to deviate from that of the parents or the average of the parents. In this study, we analyzed gene expression changes in previously reported, highly stable synthetic wheat allohexaploids that combine the D genome of Aegilops tauschii and the AB genome extracted from the natural hexaploid wheat Triticum aestivum. A comprehensive genome-wide analysis of transcriptional changes using the Affymetrix GeneChip Wheat Genome Array was conducted. Prevalence of gene expression additivity was observed where expression does not deviate from the average of the parents for 99.3% of 34,820 expressed transcripts. Moreover, nearly similar expression was observed (for 99.5% of genes) when comparing these synthetic and natural wheat allohexaploids. Such near-complete additivity has never been reported for other allopolyploids and, more interestingly, for other synthetic wheat allohexaploids that differ from the ones studied here by having the natural tetraploid Triticum turgidum as the AB genome progenitor. Our study gave insights into the dynamics of additive gene expression in the highly stable wheat allohexaploids.

Genome-wide gene expression changes in genetically stable synthetic and natural wheat allohexaploids.

*The present study aims to understand regulation of gene expression in synthetic and natural wheat (Triticum aestivum) allohexaploids, that combines the AB genome of Triticum turgidum and the D genome of Aegilops tauschii; and which we have recently characterized as genetically stable. *We conducted a comprehensive genome-wide analysis of gene expression that allowed characterization of the effect of variability of the D genome progenitor, the intergenerational stability as well as the comparison with natural wheat allohexaploid. We used the Affymetrix GeneChip Wheat Genome Array, on which 55 049 transcripts are represented. *Additive expression was shown to represent the majority of expression regulation in the synthetic allohexaploids, where expression for more than c. 93% of transcripts was equal to the mid-parent value measured from a mixture of parental RNA. This leaves c. 2000 (c. 7%) transcripts, in which expression was nonadditive. No global gene expression bias or dominance towards any of the progenitor genomes was observed whereas high intergenerational stability and low effect of the D genome progenitor variability were revealed. *Our study suggests that gene expression regulation in wheat allohexaploids is established early upon allohexaploidization and highly conserved over generations, as demonstrated by the high similarity of expression with natural wheat allohexaploids.

Newly synthesized wheat allohexaploids display progenitor-dependent meiotic stability and aneuploidy but structural genomic additivity.

To understand key mechanisms leading to stabilized allopolyploid species, we characterized the meiotic behaviour of wheat allohexaploids in relation to structural and genetic changes. For that purpose, we analysed first generations of synthetic allohexaploids obtained through interspecific hybridization, followed by spontaneous chromosome doubling, between several genotypes of Triticum turgidum and Aegilops tauschii wheat species, donors of AB and D genomes, respectively. As expected for these Ph1 (Pairing homoeologous 1) gene-carrying allopolyploids, chromosome pairing at metaphase I of meiosis essentially occurs between homologous chromosomes. However, the different synthetic allohexaploids exhibited progenitor-dependent meiotic irregularities, such as incomplete homologous pairing, resulting in univalent formation and leading to aneuploidy in the subsequent generation. Stability of the synthetic allohexaploids was shown to depend on the considered genotypes of both AB and D genome progenitors, where few combinations compare to the natural wheat allohexaploid in terms of regularity of meiosis and euploidy. Aneuploidy represents the only structural change observed in these synthetic allohexaploids, as no apparent DNA sequence elimination or rearrangement was observed when analysing euploid plants with molecular markers, developed from expressed sequence tags (ESTs) as well as simple sequence repeat (SSR) and transposable element sequences.

Homoeologous recombination within bread wheat to develop novel combinations of HMW-GS genes: transfer of the Glu-A1 locus to chromosome 1D.

In an attempt to improve the bread-making quality within hexaploid wheat by elaborating novel high-molecular weight glutenin subunits (HMW-GS) combinations useful in wheat-breeding programmes, a 1A chromosome fragment carrying the Glu-A1 locus encoding the subunit Ax2*, was translocated to the long arm of chromosome 1D. The partially isohomoeoallelic line, designated RR239, had a meiotic behaviour as regular as cv. Courtot. It was characterised using genomic in situ hybridization and microsatellite markers as well as biochemical and proteomic approaches. The translocated 1D chromosome had an interstitial 1AL segment representing in average 30% of the recombinant arm length that was confirmed by molecular analysis. The genetic length of the removed segment in chromosome 1DL was estimated to be at least 51 cM, and that of the interstitial 1AL translocation to be at least 33 cM. Proteome analysis performed on total endosperm proteins revealed variation in amounts, 8 spots and 1 spot being up- and downregulated, respectively. Quantitative variations in HMW-GS were observed for the Glu-A1 (Ax2*) and Glu-B1 (Bx7 + By8) loci in response to duplication of the Glu-A1 locus.

Development of isohomoeoallelic lines within the wheat cv. Courtot for high molecular weight glutenin subunits: transfer of the Glu-D1 locus to chromosome 1A.

Wheat quality depends on protein composition and grain protein content. High molecular weight glutenin subunits (HMW-GS) play an important role in determining the viscoelastic properties of gluten. In an attempt to improve the bread-making quality of hexaploid wheat by elaborating novel HMW-GS combinations, a fragment of wheat chromosome 1D containing the Glu-D1 locus encoding the Dx2+Dy12 subunits was translocated to the long arm of chromosome 1A using the ph1b mutation. The partially isohomoeoallelic line selected was characterized using cytogenetical and molecular approaches to assess the amount of chromatin introgressed in the translocated 1A chromosome. Triple-target genomic in situ hybridization indicated that the translocated 1A chromosome had a terminal 1D segment representing 25% of the length of the recombinant long arm. The translocation was also identified on the long arm using molecular markers, and its length was estimated with a minimum of 91 cM. Proteome analysis was performed on total endosperm proteins. Out of the 152 major spots detected, 9 spots were up-regulated and 4 spots were down-regulated. Most of these proteins were identified as alpha-, beta-, gamma-gliadins assigned to the chromosomes of homoeologous groups 1 and 6. Quantitative variations in the HMW-GS were only observed in subunit Dy12 in response to duplication of the Glu-D1 locus.

Assignment of Aegilops variabilis Eig chromosomes and translocations carrying resistance to nematodes in wheat.

The allotetraploid species Aegilops variabilis Eig (2n = 28, UUSvSv) belongs to the tribe Triticeae and is closely related to wheat. One accession, Ae. variabilis No. 1, was found to be resistant to the cereal cyst nematode (CCN) and the root-knot nematode (RKN). As the genetic variability for resistance to those two pests is limited within wheat, this accession was crossed to bread wheat. Previous work enabled the development of two addition lines and two translocation lines carrying resistance. Here, we demonstrate, using genomic in situ hybridization, that there is no U-Sv interchange in the parental accession of Ae. variabilis. However, there are multiple rearrangements in the Sv chromosomes. The Ae. variabilis chromosome carrying the CreX gene for resistance to CCN combined segments with homoeology to wheat groups 1, 2, 4, and 6. The CreX gene belongs to the group 1 part and it was likely to have been introduced into chromosome 1BL at a similar location as the previously found QTL QCre.srd-1B for CCN resistance. The second Ae. variabilis chromosome carrying CreY and Rkn2 combined segments with homoeology to wheat groups 2, 4, and 7 on its short arm and group 3 on its long arm. It was designated as 3Sv. The two genes for resistance are carried by its long arm and have been transferred to wheat chromosome 3BL through homoeologous and genetically balanced recombination. Different SSR markers present in the introgressed segments could be used in marker-assisted selection.

Attempts to induce homoeologous pairing between wheat and Agropyron cristatum genomes.

Agropyron cristatum (2n = 4x = 28, PPPP) possesses potentially valuable traits that could be used in wheat (Triticum aestivum) improvement through interspecific hybridization. Homoeologous pairing between wheat chromosomes and P chromosomes added to wheat in a set of wheat - A. cristatum addition lines was assessed. First, the Ph-suppressing effect of P chromosomes (except 7P) was analyzed. It was concluded that this system is polygenic with no major gene, and consequently, has no prospect in the transfer of alien genes from wild relatives. In a second step, the potential of the deletion ph1b of the Ph1 gene for inducing P-ABD pairing was evaluated. Allosyndetic associations between P and ABD genomes are very rare. This very low level of pairing is likely due to divergence in the repeated sequences between Agropyron and wheat genomes. Development of translocation lines using ionizing radiation seems to be a more suitable technique than homoeologous recombination to exploit the A. cristatum genome in wheat improvement.

Structure of Aegilops ventricosa chromosome 6Nv, the donor of wheat genes Yr17, Lr37, Sr38, and Cre5.

An Aegilops ventricosa Tausch (2n = 28, DvDvNvNv) subtelocentric chromosome added to wheat (Triticum aestivum L.) in a disomic addition line was found to carry the genes for resistance Yr17, Lr37, Sr38, and Cre5 already transferred onto chromosome 2AS of the wheat line VPM1. Previous works demonstrated that this Ae. ventricosa chromosome is translocated with respect to the standard wheat genome. The present investigations showed that this chromosome pre-existed in Ae. ventricosa and contains only chromatin specific to the N genome. Using biochemical markers and suitable cytogenetic materials including the monoisosomic addition line for the translocated long arm (6NvL-2NvS), its structure was defined as being 6NvSdel.6NvL-2NvS. It consists of a segment of the short arm 2Nv, containing the resistance genes, attached to a group 6 chromosome lacking a distal part of its short arm. The 2 re arrangements could already be present in Aegilops uniaristata Vis. (2n = 14, NN), the source of the Nv genome of Ae. ventricosa.

Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot.

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.